Abstract

In recent years, a number of tools have become available for remotely activating neural circuits in Drosophila. Despite widespread and growing use, very little work has been done to characterize exactly how these tools affect activity in identified fly neurons. Using the GAL4-UAS system, we expressed blue light-gated Channelrhodopsin-2 (ChR2) and a mutated form of ChR2 (H134R-ChR2) in motor and sensory neurons of the Drosophila third-instar locomotor circuit. Neurons expressing H134R-ChR2 show enhanced responses to blue light pulses and less spike frequency adaptation than neurons expressing ChR2. Although H134R-ChR2 was more effective at manipulating behavior than ChR2, the behavioral consequences of firing rate adaptation were different in sensory and motor neurons. For comparison, we examined the effects of ectopic expression of the warmth-activated cation channel Drosophila TRPA1 (dTRPA1). When dTRPA1 was expressed in larval motor neurons, heat ramps from 21 to 27 degrees C evoked tonic spiking at approximately 25 degrees C that showed little adaptation over many minutes. dTRPA1 activation had stronger and longer-lasting effects on behavior than ChR2 variants. These results suggest that dTRPA1 may be particularly useful for researchers interested in activating fly neural circuits over long time scales. Overall, this work suggests that understanding the cellular effects of these genetic tools and their temporal dynamics is important for the design and interpretation of behavioral experiments.

Thresholds for evoking single EJPs at the m6 neuromuscular junction are different for ChR2 and H134R-ChR2. A: single EJPs evoked by 10-ms pulses of varying intensity. Blue arrows denote light pulses. Note that EJPs are evoked at a lower light intensity in the H134R-ChR2–expressing preparation. B: percent of responses as a function of light intensity and duration in the 2 ChR2 variants. Pooled data shown as means ± SE. *P < 0.05, Student's t-test.

H134R-ChR2–expressing animals show less spike frequency adaptation than ChR2-expressing animals. A: light-evoked large EJPs in m6 in response to constant blue light stimulation of animals expressing ChR2 (top) or H134R-ChR2 (bottom) in motor neurons under control of OK371-GAL4. Instantaneous EJP frequency is shown above each trace. Note dashed line at 20 Hz in both panels. B: pooled data showing the decay of EJP frequency (means ± SE) as a function of genotype and stimulation rhythmicity. *Significant differences from ChR2 constant light data at each time point (P < 0.05, ANOVA with Bonferroni post hoc test); color of asterisk indicates which data set is being compared. Blue light pulses were 470 nm and 300 μW/mm2 for all experiments. n for each data set is indicated on the histogram bar.

Acute activation of motor neurons with ChR2 and H134R-ChR2 affects locomotion. A: larval locomotor response to constant illumination in animals expressing ChR2 in motor neurons (blue) and in genetic controls (black and gray). Crawling speeds of ChR2 animals were not significantly different from both controls at any time point. B: same experiment as in A, but with H134R-ChR2 expressed in motor neurons (red). Red asterisks indicate times when H134R-ChR2 expressing animals crawl significantly more slowly than both controls (P < 0.05 for t = 10–70 s). Pink # symbols indicate times when H134R-ChR2 animals crawl slower than animals expressing ChR2 (P < 0.05 for t = 20–130 s). C: response to rhythmic (500 ms on, 500 ms off) illumination in ChR2-expressing motor neurons (light blue) and in genetic controls (black and gray). There were no significant differences in crawling speeds between experimental and control animals throughout the trial. D: same experiment as in C, but with H134R-ChR2 expressed in motor neurons (orange). Experimental animals were not different from both controls at any point. In all experiments, crawling speeds were binned to yield average speed for 10-s intervals. Note that in A and B and again in C and D, GAL4 control data are shown twice, to facilitate comparison. Blue bars represent duration of stimulus. Data are presented as means ± SE. *P < 0.05 (ANOVA with Bonferroni post hoc test).